Identification And Analysis Of Gastrodia Elata MiRNA And Preliminary Studies On Its Cross-kingdom Regulation

Gastrodia elata is one of precious Chinese herbal medicines,which has a variety of functions such as anticonvulsant effect,neuroprotection,anti-inflammatory and immune regulation.At present,the research on Gastrodia elata mainly focuses on the pharmacological research and product development on the crude extracts or natural chemical components of Gastrodia elata,but there is no report on microRNA in Gastrodia elata.MicroRNAs(miRNAs)are a type of small,non-coding,19-24 nt biological RNA molecules which is a close relationship between miRNAs and human disease activities.The miRNAs can interact with target mRNAs through pairing to degrade target mRNA or inhibit their translation,playing an important role in cell proliferation,differentiation and senescence by regulating key factors in a variety of signaling pathways.In the present work,we studied the role of Gastrodia elata in the process of inflammation from the perspective of cross-kingdom miRNA,and lay a foundation for revealing its mechanism of anti-inflammatory and immunity at the molecular level.It provides new evidence for the regulation of miRNAs and provides data support for the subsequent study of Gastrodia elata miRNAs in medicine and pharmacy.First,Gastrodia elata small RNAs were sequenced by Illumina high-throughput sequencing.9,798,753,10,713,576,11,916,192 and 11,229,380 raw data were obtained from the four tissues(“jianma” Bud,“hema” Bud,“jianma” Tuber and“hema” Tuber)of Gastrodia elata,respectively.After removing low quality,linker and decontamination,9,666,044,10,508,310,11,763,094 and 11,074,999 clean reads were obtained,respectively.And then the length of the clean sequences was counted statistically,and these clean reads are mainly miRNA and siRNAs,suggesting that these small RNAs might play an important role in regulating gene expression.From these reads,5718 known miRNAs were found,of which 52 were new miRNAs.The relative expression level of 10 seleted candidate miRNAs(Gas-miR159,Gas-miR6478,Gas-miR148a-3p,Gas-miR99,Gas-miR143-3p,Gas-miR319 f,Gas-miR396 e,Gas-miR26 a,Gas-mi R01 and Gas-miR02)in different Gastrodia elata tissues was further validated by qRT-PCR,and the results were consistent with the results of high throughput sequencing.Secondly,qRT-PCR was used to detect the relative expression levels of the above ten seleted miRNAs in the different developmental stages of Gastrodia elata,such as,seeds,“mima”,“baima” and “jianma”.The results showed that the expression level of Gas-miR319 f was similar in seed,“mima” and “baima”.However,the expression level was rapidly increased to several tens of times in “jianma” period.TargetScan and RNAhybrid software were used to predict the target gene for Gas-miR01 and Gas-miR02 in the human genome.KEGG analysis showed that Gas-miR01 and Gas-miR02 could target three key inflammation-related genes A20,TNF-? and I?B? on NF-?B signaling pathway,suggesting that these two Gastrodia elata-specific miRNA molecules might regulate the key genes in NF-?B signaling pathway at a cross-kingdom manner,and be involved in human immune and inflammatory response and other physiological processes.Then,the relative levels of Gas-miR01 and Gas-miR02 were identified by qRT-PCR in each tissue of experimental mice that were orally administered with decoction,gastrodia powder and total RNAs.The results showed that compared with the control group,the levels of Gas-miR01 and Gas-miR02 significantly increased in all tissues in the experimental group,demonstrating that Gas-miR01 and Gas-mi R02 can enter the mouse through the intestine,which provides an important basis for the study of the efficacy of small RNA in Gastrodia elata.Finally,Gas-miR01 mimics,Gas-miR02 mimics and miRNA negative control were transfected into 293 T cells,respectively.Western Blotting showed that both Gas-miR01 mimics and Gas-miR02 mimics could down-regulate the key protein A20 in NF-?B signaling pathway.A20 is a NF-?B-dependent protein.When NF-?B is activated in a large amount,A20 can be phosphorylated by IKK? and inhibit the activation of NF-?B,which plays a role in feedback regulation.This discovery provides a new path for the further development of Gastrodia elata anti-inflammatory drugs.It also provides a scientific basis for further research on the mechanism of miRNA action of Gastrodia elata.